Enzymes that catalyse protein modification are central players in signalling pathways. For such enzymes, substrate recognition can involve binding to short, linear peptides that include the modified residue or are separate “docking sites”. This report demonstrates the power of HiTrap™ NHS-activated columns in identifying and analysing such interactions. We prepared affinity columns using HiTrap NHS-activated columns and a pentapeptide sequence (NWETF) present at the extreme carboxyl terminus of some bacterial chemoreceptors. This affinity column enabled the demonstration that the pentapeptide serves as a docking site on chemoreceptors for both the methyltransferase and methylesterase. The column was also used to isolate the methylesterase to high purity (99.4%) and identify the pentapeptide-binding site on the methylesterase.

Introduction
Bacterial chemotaxis is mediated by transmembrane receptor proteins with periplasmic domains that bind attractants and cytoplasmic domains that control the activity of a histidine kinase. Chemotaxis requires gradient sensing and sensory adaptation, both of which are accomplished via methylation and demethylation at specific glutamyl residues in the receptor cytoplasmic domains. These reactions are catalysed by the dedicated methyltransferase, CheR, and the dedicated methylesterase, CheB (Fig 1).

Recent work has shown that a pentapeptide sequence, asn-trp-glu-thr-phe (NWETF in the one-letter code), present at the extreme carboxyl terminus of some chemoreceptors (the “high-abundance” receptors), provides a docking site for the methyltransferase (1) and greatly enhances methylation of receptors on which the sequence is present (2–5). The methyltransferase binds the docking site through a subdomain not present on the methylesterase. Yet, we discovered that chemoreceptors lacking the carboxyl-terminal pentapeptide were not only poor substrates for methylation, but also exhibited greatly reduced rates of demethylation in vitro (6).

A tantalizing explanation for the dependence of demethylation, as well as methylation, on the pentapeptide was that the sequence served as a docking site not only for the methyltransferase but also for the methylesterase. We tested this hypothesis by creating an affinity column in which the pentapeptide was coupled to a HiTrap NHS-activated column by its single amino group (at the amino terminus), and by examining retention and specific elution of the methylesterase.

Materials and methods
The pentapeptide NWETF was synthesized by solid-phase synthesis using Fmoc chemistry and coupled specifically at its amino terminus using EDC-NHS coupling chemistry.

Coupling was done at room temperature for 40 min using 1 ml (analytical) or 5 ml (preparative) HiTrap NHS-activated columns (Amersham Pharmacia Biotech) and the protocol provided with the columns. HiTrap NHS-activated columns were connected to peristaltic pump P1 (Amersham Pharmacia Biotech). For coupling, NWETF was dissolved in 0.2 M NaHCO₃, 0.5 M NaCl (pH 8.3) at 5 mg/ml without any additional purification after synthesis.

After washing and deactivation, the column was equilibrated with 50 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 2 mM dithiothreitol, and 10% glycerol (TEDG). Cell lysate (~ 2 mg protein/ml) containing CheR or CheB produced from an induced, plasmid-borne gene (6), was applied to the column, followed by two column volumes of TEDG and three column volumes of TEDG containing 5 mg/ml pentapeptide. Each fraction was analysed by SDS-PAGE.

For purification of preparative amounts of CheB, 5 ml columns were eluted with 2 M NaCl instead of pentapeptide, yielding a protein over 99% pure as estimated by Coomassie Blue staining of overloaded SDS polyacrylamide gels. The 21 000 M_r species corresponding to the proteolytically produced, catalytic domain of CheB was only ~ 0.02% the intensity of intact CheB.

The interaction of methylesterase with the pentapeptide was also utilized for enzyme purification. A 5 ml HiTrap affinity column, in combination with elution by high salt instead of pentapeptide, provided a rapid, one-step isolation that produced high purity (99.4%) enzyme. This preparation contained minimal amounts of the proteolytically produced catalytic domain of the methylesterase, a major contaminant in conventional purifications (7, 8).

Recently we used the pentapeptide column to identify the segment of the methylesterase that interacts with the pentapeptide. The pattern of elution and retention of proteolytic fragments implicated a small segment (~ 20 amino acids) of one of the two esterase domains as the site of pentapeptide binding (data not shown).

The immobilized pentapeptide affinity column has proved very effective for identification, purification, and analysis of enzymes that recognize a short peptide docking site. It is noteworthy that our affinity purification greatly reduced contamination by a proteolytic fragment that complicated conventional purification, and that a binding site could be localized by analysis of enzyme fragments. Peptide coupling to an NHS-activated column has the potential of becoming a general strategy for identifying, purifying, and analysing proteins that recognize linear docking sites.

References
1.	Wu, J. et al., Biochemistry 35, 4984–4993 (1996).
2.	Li, J. et al., Biochemistry 36, 11851–11857 (1997).
3.	Le Moual, H. et al., Biochemistry 36, 13441–13448 (1997).
4.	Barnakov, A. N. et al., J. Bacteriol. 180, 6713–6718 (1998).
5.	Feng, X. et al., J. Bacteriol. 181, 3164–3171 (1999).
6.	Barnakov, A. N. et al., Proc. Natl. Acad. Sci. USA 96, 10667–10672 (1999).
7.	Simms, S. A. et al., J. Biol. Chem. 280, 10161–10168 (1985).
8.	Anand, G. S. et al., Biochemistry 37, 14038–14047 (1998).

Acknowledgement
We thank Bryan Beel for a figure on which Fig 1 is based.
Supported by Grant GM29963 from the NIH.

Ordering information
HiTrap NHS-activated, 1 ml	5 x 1 ml	17-0716-01
HiTrap NHS-activated, 5 ml	1 x 5ml	17-0717-01